Knowledge is very limited with respect to how facultative pathogens, i.e., pathogens that spend portions of their life cycles both inside and outside of the human body, adapt to drastically changing conditions when passing from a human host into an environmental reservoir. Advances in understanding of the basic mechanisms of adaptation during this transition are needed to provide new molecular targets and strategies for limiting transmission, dissemination and environmental persistence, and thus reduce the large medical burden imposed by this diverse group of pathogens. In the facultative, water-borne pathogen Vibrio cholerae (the causative agent of cholera), signaling by the intracellular secondary messenger molecule cyclic diguanylate (c-di-GMP) leads to the repression of virulence genes, and at the same time, the induction of environmental survival genes. At a late stage of infection, just prior to excretion of V. cholerae in watery stool, expression of three genes that encode c-di-GMP synthetases is induced, leading to the hypothesize that c-di-GMP mediates the transition from a state of virulence to one of environmental suitability. In this project the role of c-di- GMP in the biology of V. cholerae late in infection and in the process of dissemination will be determined using genetic and biochemical methods, including some novel methods created for these studies. In addition, the roles of a family of five c-di-GMP-binding proteins in transducing the c-di- GMP signal into biological changes will be determined. It is anticipated that this work will provide a working model of the central regulatory pathway in V. cholerae that represses virulence gene expression and induces expression of genes important for dissemination. It is also anticipated that these studies will have application to a broad range of bacterial pathogens since most contain multiple c-di-GMP synthetic, degradative and sensory proteins.